Field of the Invention
Embodiments of the present invention relate in general to methods and compositions for assembling nucleic acid sequences.
Description of Related Art
The development of inexpensive, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology (Carr & Church (2009) Nat. Biotechnol. 27:1151). Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis (Tian et al. (2009) Mol. BioSyst. 5:714). Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale microchip use have been largely unsuccessful due to the high error rates and complexity of the oligonucleotide mixtures (Tian et al. (2004) Nature 432:1050; Richmond et al. (2004) Nucleic Acids Res. 32:5011; Zhou et al. (2004) Nucleic Acids Res. 32:5409).
The synthesis of novel DNA encoding regulatory elements, genes, pathways, and entire genomes provides powerful ways to both test biological hypotheses as well as harness biology for humankind's use. For example, since the initial use of oligonucleotides in deciphering the genetic code, DNA synthesis has engendered tremendous progress in biology with the recent complete synthesis of a viable bacterial genome (Nirenberg et al. (1961) Proc. Natl. Acad. Sci. USA 47:1588; Söll et al. (1965) Proc. Natl. Acad. Sci. USA 54:1378; Gibson et al. (2010) Science 329:52). Currently, almost all DNA synthesis relies on the use of phosphoramidite chemistry on controlled-pore glass (CPG) substrates. CPG oligonucleotides synthesized in this manner are effectively limited to approximately 100 bases by the yield and accuracy of the process. Thus, the synthesis of gene-sized fragments relies on assembling many oligonucleotides together using a variety of techniques termed gene synthesis (Tian (2009) (supra); Gibson (supra); Gibson (2009) Nucleic Acids Res. 37:6984; Li & Elledge (2007) Nat. Methods 4:251; Bang & Church (2008) Nat. Methods 5:37; Shao et al. (2009) Nucleic Acids Res. 37:e16).
The price of gene synthesis has reduced drastically over the last decade as the process has become increasingly industrialized. However, the current commercial price of gene synthesis, approximately $0.40-1.00/bp, has begun to approach the relatively stable cost of the CPG oligonucleotide precursors (approximately $0.10-0.20/bp) (Carr (supra)). At these prices, the construction of large gene libraries and synthetic genomes is out of reach to most. To achieve further cost reductions, many current efforts focus on smaller volume synthesis of oligonucleotides in order to minimize reagent costs. For example, microfluidic oligonucleotide synthesis can reduce reagent cost by an order of magnitude (Lee et al. (2010) Nucleic Acids Res. 38:2514).
Another route is to harness existing DNA microchips, which can produce up to a million different oligonucleotides on a single chip, as a source of DNA for gene synthesis. Previous efforts have demonstrated the ability to synthesize genes from DNA microchips. Tian et al. described the assembly of 14.6 kb of novel DNA from 292 oligonucleotides synthesized on an Atactic/Xeotron chip (Tian (2004) (supra)). The process involved using 584 short oligonucleotides synthesized on the same chip for hybridization-based error correction. The resulting error rates were approximately 1/160 basepairs (bp) before error correction and approximately 1/1400 bp after. Using similar chips, Zhou et al. constructed approximately 12 genes with an error rate as low as 1/625 bp (Zhou (supra)). Richardson et al. showed the assembly of an 180 bp construct from eight oligonucleotides synthesized on a microarray using maskless photolithographic deprotection (Nimblegen) (Richmond (supra)). Though the error rates were not determined in that study, a follow-up construction of a 742 bp green fluorescent protein (GFP) sequence using the same process showed an error rate of 1/20 bp-1/70 bp (Kim et al. (2006) Microelectronic Eng. 83:1613). These approaches have thus far failed to scale for at least two reasons. First, the error rates of chip-based oligonucleotides from DNA microchips are higher than traditional column-synthesized oligonucleotides. Second, the assembly of gene fragments becomes increasingly difficult as the diversity of the oligonucleotide mixture becomes larger.